Viral SAM-binding proteins nsp14 and NP868R reprogram ATG4A-dependent autophagy from antiviral LC3B activity to GABARAP-mediated mitophagy

Members of the mammalian Atg8-protein family (ATG8), including the MAP1LC3/LC3 and GABARAP subfamilies, play essential roles in selective macroautophagy/autophagy. However, their functional distinctions during viral infection remain poorly understood. Here, we show that S-adenosyl-L-methionine (SAM)-binding viral proteins, such as nsp14 from coronavirus and NP868R from African swine fever virus (ASFV), reprogram autophagy by shifting antiviral LC3B activity toward GABARAP-mediated mitophagy in an ATG4A-dependent manner. Mechanistically, the SAM-binding motif allows these viral proteins to stabilize <i>ATG4A</i> mRNA, thereby increasing ATG4A expression and redirecting autophagic flux from LC3B-mediated virophagy to GABARAP-dependent mitophagy. This shift suppresses innate immune responses by targeting both MAVS-dependent interferon signaling and virophagy, ultimately enhancing viral replication. Collectively, our findings uncover a previously unrecognized immune evasion strategy in which SAM-binding viral proteins rewire autophagy from antiviral to proviral pathways.<b>Abbreviation:</b> ACTB: actin beta; ATG: autophagy related genes; ASFV: African swine fever virus; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CQ: chloroquine; CS: citrate synthase; ExoN: exoribonuclease; GABARAP: GABA type A receptor-associated protein; IFN: type I interferon; IFNB: interferon beta; IPEC-J2: intestinal porcine epithelial cell line-J2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MT-CO2/COX2: mitochondrially encoded cytochrome c oxidase II; nsp14: nonstructural protein 14; OPTN: optineurin; PEDV: porcine epidemic diarrhea virus; RNMT/N7-MTases: RNA guanine-7 methyltransferase; SAM: S-adenosyl-L-methionine; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TCID₅₀: 50% tissue culture infective dose; TOMM70: translocase of outer mitochondrial membrane 70; TOMM20: translocase of outer mitochondrial membrane 20; WT: wild-type.